Photoinduced electron transfer (pet) primer for nucleic acid amplification

ABSTRACT

This application provides photoinduced electron transfer (PET) nucleic acid molecules that can be used detect and amplify nucleic acid molecules, such as target nucleic acid molecules. These PET tags can be attached to the 5′-end of a target sequence-specific primer, thereby generating a PET primer. In particular examples, a PET tag includes a 5′-labeled nucleotide that can be quenched by at least two consecutive Gs within the tag sequence, and is unquenched when the PET tag hybridizes with its complementary nucleic acid molecule. Also disclosed are methods of using PET primers in nucleic acid amplification, such as real-time PCR.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/743,607 filed on May 19, 2010, which is the U.S. National Stage ofInternational Application No. PCT/US2008/084347, filed Nov. 21, 2008,which claims the benefit of the earlier filing date of U.S. ProvisionalApplication No. 60/989,768, filed on Nov. 21, 2007, which areincorporated herein by reference.

FIELD

The present disclosure relates to labeled nucleic acid primers andmethods of their use, for example to detect or amplify a target nucleicacid molecule.

BACKGROUND

The real-time polymerase chain reaction (PCR) is currently used as adiagnostic tool in clinical applications, and can be used to obtainquantitative results. The chemistry of real-time PCR is based onmonitoring fluorescence at every cycle at a set temperature thatfacilitates calculating the kinetics of the product formed andperforming melting curve analysis to identify formation of the specificproduct. Fluorescence is usually monitored using an optical device tocollect the data at specific excitation and emission wavelengths for theparticular fluorophore present in the sample.

One method used to monitor nucleic acid amplification is the addition ofintercalating dyes, such as SYBR Green I dye (Ririe et al., Anal.Biochem. 245:154-60, 1997) and LCGreen (Wittwer et al., Clin. Chem.49:853-60, 2003) during PCR. During amplification, these dyes areexcited with the appropriate wavelength of light, inducing fluorescencewhen the dye intercalates into a DNA double helix. However, this methoddoes not allow for multiplex reactions.

Specificity can be increased by using a labeled sequence-specific probe.Several of such methods are currently available for performing real-timePCR, such as TaqMan® probes (Lee et al., Nucleic Acids Res. 21:3761-6,1993); molecular beacons (Tyagi and Kramer, Nat. Biotechnol. 14:303-8,1996); self-probing amplicons (scorpions) (Whitcombe et al., Nat.Biotechnol. 17:804-7, 1999); Amplisensor (Chen et al., Appl. Environ.Microbiol. 64:4210-6, 1998); Amplifluor (Nazarenko et al., Nucleic AcidsRes. 25:2516-21, 1997 and U.S. Pat. No. 6,117,635); displacementhybridization probes (Li et al., Nucleic Acids Res. 30:E5, 2002);DzyNA-PCR (Todd et al., Clin. Chem. 46:625-30, 2000); fluorescentrestriction enzyme detection (Cairns et al. Biochem. Biophys. Res.Commun. 318:684-90, 2004); and adjacent hybridization probes (Wittwer etal., Biotechniques 22:130-1, 134-8, 1997).

Some currently available labeled primers can have a secondary structurethat is complex and in some instances must be synthesized usingspecialized procedures. For example, LUX™ primers (Invitrogen Corp.) arefluorescently labeled on the 3′-end and have a stem-loop structure thatmust be denatured for the primer to work efficiently (especially forreverse transcription). The design of the LUX™ primer is also atime-consuming step, which requires specific software.

Several publications disclose probes that contain only one fluorophorefor use in detecting the presence of a particular nucleic acid [forexample see U.S. Pat. No. 6,699,661; U.S. Pat. No. 6,495,326; and U.S.Pat. No. 6,492,121 (all to Kurane et al.); U.S. Pat. No. 6,635,427(Wittwer et al.); Kurata et al. (Nucl. Acids Res. 29:E34, 2001);Torimura et al. (Analyt. Sci. 17:155-60, 2001); and Crockett et al.(Analyt. Biochem. 290:89-97, 2001)]. In these examples, the fluorescentsignal is either enhanced or quenched in the presence of the targetnucleic acid sequence, depending on the particular design of the probe.In most cases, the labeled primer specifically hybridizes to the targetnucleic acid sequence. Similarly, Tam-Chang (Analyt. Biochem.366:126-130, 2007) discloses a multi-probe universal reporter systemcontaining a signal that is enhanced only after sequence-specifichybridization of one of the probes. Guo and Milewicz (Biotech. Lett.25:2079-83, 2003) disclose universal fluorescent tag primers labeled onthe 5′ end that are not sequence specific. The labeled fluorescent taguniversal primer, in combination with two sequence-specific primers, areuse to amplify a target nucleic acid sequence.

Yamane (Nucl. Acids Res. 30:E97, 2002) discloses a MagniProbe that hasan internal fluorophore and an internal intercalator. The fluorescenceis quenched by the intercalator in the absence of a target sequence.Upon hybridization with the target sequence, the probe emitsfluorescence due to the interference in quenching by intercalation.

Nazarenko et al. (Nucl. Acids Res. 30:E37, 2002) disclose a probe with asingle fluorophore near the 3′ end (but no quencher), and addition of5-7 base pairs to the 5′ end of the sequence-specific probe, wherein thesignal from the fluorophore is increased in the presence of the targetsequence.

SUMMARY

The present application relates to novel photoinduced electron transfer(PET) nucleic acid molecules (also referred to herein as PET tags). Alsoprovided are methods for using the PET tags, for example in assessingthe progress of PCR, such as real time PCR, or for assessing theprogress of melting duplex DNA, such as an amplicon. The novel PET tagsinclude a 5′-end-labeled nucleotide, and can further include atarget-specific sequence at the 3′-end of the PET tag, therebygenerating a labeled sequence-specific primer sequence (also referred toherein as a PET primer). Thus, methods are provided for generatinglabeled sequence-specific primers, by adding or attaching a primerspecific for a target sequence to a labeled PET tag. In the absence ofhybridization of the PET tag to is complementary sequence, thedetectable signal is altered (such as quenched) by at least twoconsecutive G nucleotides (or other nucleotides that can permitquenching of the signal from the 5′-end labeled nucleotide, such as isoCand isoG) brought into proximity to the label due to a stem-loop thatincludes complimentary nucleotide sequences. When the PET tag hybridizesto its complement sequence (e.g., when present in an amplicon), thestem-loop becomes linear, thereby increasing the distance between thelabel and the at least two consecutive G nucleotides (or isoC or isoG)and alternating the signal from the label (such as increasing thedetectable signal).

In particular examples, the disclosed PET nucleic acid molecules includea 5′-end-labeled nucleotide, a stem-loop, and at least two consecutive Gnucleotides (or other nucleotides that can permit quenching of the labelon the 5′-end nucleotide, such as isoC and isoG), wherein the stem-loopincludes complimentary nucleotide sequences in the stem portion, therebybringing the label on the 5′-end-labeled nucleotide and the at least twoconsecutive G nucleotides into proximity, thereby changing (such asquenching) a detectable signal from the 5′-end-labeled nucleotide. Atarget- or sequence-specific primer can be attached to the 3′-end of thePET tag. In some examples, the at least two consecutive G nucleotidesadjacent to the stem-loop of the PET tag can be the first twonucleotides at the 5′-end of the target- or sequence-specific primer. Insome examples, there are one or more nucleotides (or other spacer)between the sequence-specific primer and the at least two consecutive Gnucleotides of the PET tag, such as 1-10 nucleotides. When the PET taghybridizes with its complementary sequence, the 5′-end-labelednucleotide is no longer in close proximity to the at least twoconsecutive G nucleotides, thereby changing the detectable signal fromthe label (such as increasing the detectable signal).

In particular examples, a PET tag includes the sequence5′-X₁X_(2(a))X₃X_(4(a))G_(x)-3′ (SEQ ID NO: 1), wherein X₁ is the 5′-endlabeled nucleotide, wherein X₂ and X₄ include complimentary nucleotidesequences of length a, wherein X₃ includes the loop of the stem-loop,wherein G_(x) includes the at least two consecutive G nucleotides. Forexample, the PET tag can include the sequence5′-X₁X_(2(a))X₃X_(4(a))G_(x)X_(5(n))-3′ (SEQ ID NO: 2), wherein X₅includes “n” number of nucleotides, for example n can be zero or morenucleotides (such as one or more nucleotides, for example 1-5nucleotides). In some examples, X₁ is any nucleotide, but in someexamples, X₁ is not G. In particular examples, X₃ is a trinucleotidesequence, such TAA, ATA, AAT, TTA, TAT, ATT, TTT or AAA. In one example,a PET tag consists of the sequence 5′-X₁X_(2(a))X₃X_(4(a))G_(x)-3′ (SEQID NO: 1) and has a sequence-specific primer (e.g., a primer thatspecifically hybridizes to a target nucleic acid sequence) attached atits 5′-end to G_(x). In another example, a PET tag consists of thesequence 5′-X₁X_(2(a))X₃X_(4(a))-3′ (SEQ ID NO: 3) and hassequence-specific primer (e.g., a primer that specifically hybridizes toa target nucleic acid sequence) with at least two consecutive Gnucleotides on its 5′-end attached at its 5′-end to X_(4(a)) of the PETtag. Such molecules can be referred to as a labeled sequence-specificprimer or a PET primer.

Although particular exemplary PET tags and primers are disclosed herein(for example SEQ ID NOS: 1-3, 10-27, 33 and 35-36), the presentapplication is not limited to these particular sequences.

The signal from the label changes when the PET tag or primer ishybridized to its complementary sequence, for example when it becomesincorporated into an amplicon. The change in the signal can be anincrease or a decrease, for example relative to a signal in the absenceof the complementary sequence. The resulting change in detectable signalis proportional to the amount of amplicon produced and therefore occursonly when a complimentary stand is synthesized. The signal can bedetected by a variety of devices, such as fluorescent microtiter platereaders, spectrofluorometers, fluorescent imaging systems, and real-timePCR instruments.

Any label can be used, such as a fluorophore, for example6-carboxyfluorescein (6-FAM). In particular examples, a label is onewhose signal is significantly decreased in the presence of guanosine,isoG or isoC, such as the ability to quench fluorescence. For example,the nucleotide guanosine can quench a variety of fluorophores, such as6-FAM. Thus in some examples the label is one that can be quenched byguanosine.

Ideally, a PET tag does not recognize and hybridize to a target nucleicacid sequence in the absence of a sequence-specific primer attached tothe 3′-end of the PET tag. For example, if the target nucleic acidsequence is a human p53 sequence, the PET tag does not substantiallyhybridize to a human p53 sequence. In particular examples, the PET tagalone does not hybridize with a target nucleic acid sequence undermoderately stringent or highly stringent hybridization conditions.

The disclosed PET tags can be used to label any sequence-specific primerwithout significantly affecting the sensitivity of the amplificationreaction. Ideally, a sequence-specific primer specifically recognizes atarget nucleic acid sequence. For example, if the target sequence is ahuman p53 sequence, the sequence-specific primer can substantiallyhybridize to the p53 sequence, but the PET tag does not substantiallyhybridize to the p53 sequence. In some examples, a sequence-specificprimer can hybridize with a target nucleic acid sequence undermoderately stringent or highly stringent hybridization conditions.

A PET tag can be attached via its 3′-end (e.g., G_(x) of SEQ ID NO: 1,X_(5(n)) of SEQ ID NO: 2, or X_(4(a)) of SEQ ID NO: 3) to the 5′-end ofa forward primer or a reverse primer specific for the target nucleicacid sequence of interest, thereby generating a labeled forward orlabeled reverse primer. The resulting labeled forward or labeled reverseprimer can be used to amplify the appropriate target nucleic acid, forexample using real-time PCR, resulting in the formation of ampliconproducts. The method can further include quantifying an amount of targetnucleic acid sequence present in a sample.

Also provided by the present disclosure are kits that include one ormore PET nucleic acid molecules of the present disclosure. The kits canfurther include a ligase to permit joining of the 3′-end of a PET tag tothe 5′-end of a target sequence-specific forward or reverse primer. Insome examples, the kit includes one or more sequence-specific forward orreverse primers, such as primers that recognize and can be used toamplify a target sequence of interest. In a specific example, thesequence-specific forward or reverse primer hybridizes specifically to apathogen's nucleic acid sequence, such as a viral, bacterial, parasitic,or fungal nucleic acid sequence. In another specific example, thesequence-specific forward or reverse primer hybridizes specifically to ahuman nucleic acid sequence, such as a sequence associated with adisease (such as cancer or a hereditary disorder).

Arrays, such as a DNA microarray, that include one or more of thedisclosed PET nucleic acid molecules are encompassed by this disclosure.Such arrays can be used to determine whether a desired target sequenceis present, such as in a sample. The disclosed PET primers can behybridized to a target nucleic acid sequence attached to the array (forexample resulting in fluorescence). In other examples, one or more ofthe disclosed PET tags or primers are attached to the array.

The disclosed PET tags, for example when attached to a sequence that canhybridize to a target sequence (and thereby producing a PET primer),provide an approach to detect, and in some examples further quantify, atarget nucleic acid. Use of the PET primers is shown herein to provide ahighly sensitive detection method, which permits detection of smallquantities of target nucleic acid molecule, such as DNA. For example,the present disclosure provides methods of detecting a target nucleicacid molecule. The method can include incubating a sample containingnucleic acids (such as DNA or RNA) with a PET tag which is linked to aforward or a reverse target sequence specific primer, and with thecorresponding forward or reverse target sequence specific primer notcontaining the PET tag. The sample and labeled forward primer andreverse primer not containing the PET tag, or forward primer notcontaining the PET tag and labeled reverse primers are incubated underconditions sufficient to permit amplification of the target nucleicacid. A change in signal from the label on the resulting PET primer ismonitored, wherein a change in signal (such as an increase or decreasein signal), indicates the presence of the target nucleic acid sequence.In particular examples, both the forward and reverse target sequencespecific primers contain a PET tag.

In some examples, the change in signal is monitored during theamplification reaction, for example in real time as the amplicons areformed. In other or additional examples, the change in signal ismonitored after the amplification, for example by exposing the resultingamplicons to increased temperature to generate a melting curve. Meltingcurve analysis can be used to confirm the presence of a target nucleicacid, and can also be used to distinguish polymorphisms in amplicons.

Those skilled in the art will appreciate that the disclosed isolatednucleic acid molecules and methods can be used to amplify two or moredifferent target nucleic acid molecules (such as at least 2, at least 3,at least 4, or even at least 5 different nucleic acid sequences) in thesame amplification reaction. In particular examples, two or moredifferent PET primers, each containing a different fluorophore, areused. In other examples, the same PET tag and label are attached to atleast two different sequence-specific primers, wherein the resultingamplicons are differentiated, for example by using melting curveanalysis. In yet other examples, combinations of the same PET tagsequence and label or different PET tag sequences and labels are used.

The foregoing and other objects and features of the disclosure willbecome more apparent from the following detailed description, whichproceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B are schematic drawings showing exemplary PET tags 10 inthe non-hybridized configuration.

FIG. 2 is a schematic drawing showing an exemplary PET tag 10 in thehybridized configuration during or after nucleic acid amplification(e.g., when part of an amplicon).

FIGS. 3A and B are schematic drawings showing exemplary PET tags 10ligated or synthesized at the 3′-end to the 5′-end of asequence-specific primer 30 to generate a labeled sequence-specificprimer (or PET primer) 32 which can be used in the methods disclosedherein. These drawings generally show the PET primer as it would look aspart of an amplicon.

FIG. 4 is a graph of the quantitative PET PCR assay.

FIG. 5 is a logarithmic plot of the PET PCR assay data.

FIG. 6 is a graph of the TaqMan™ comparison assay.

FIG. 7 is a logarithmic plot of the TaqMan™ comparison assay data.

FIG. 8 is a graph of a melting curve analysis of the PET PCRamplification products.

FIG. 9 is a graph showing an increase in detectable FAM signal duringamplification of a target sequence using PET primers containingdifferent numbers of Gs at the 3′-end of the PET tag (1=no Gs, 2=1 G,3=2 Gs).

FIGS. 10A and 10B are graphs showing an increase in detectable (A) FAMand (B) HEX signal during amplification of a target sequence using (A)FAM-labeled PET tags attached to the forward primer and (B) HEX-labeledPET tags attached to the reverse primer. A Cy5® dye-labeled TaqMan®probe was used in these reactions, but the fluorescence data is shown inFIG. 10E.

FIGS. 10C and 10D are graphs showing an increase in detectable (C) FAMand (D) HEX signal during amplification of a target sequence usingFAM-labeled PET tags attached to the reverse primer and (D) HEX-labeledPET tags attached to the forward primer. A Cy5® dye-labeled TaqMan®probe was used in these reactions, but the fluorescence data is shown inFIG. 10F.

FIGS. 10E and 10F are graphs showing an increase in detectable Cy5® dyesignal from TaqMan® probes during amplification of a target sequenceusing the (E) FAM-forward primer and HEX-reverse primer described inFIGS. 10A and 10B or (F) HEX-forward primer and FAM-reverse primerdescribed in FIGS. 10C and 10D.

SEQUENCE LISTING

The nucleotide sequences of the nucleic acids described herein are shownusing standard letter abbreviations for nucleotide bases. Only onestrand of each nucleic acid sequence is shown, but the complementarystrand is understood as included by any reference to the displayedstrand.

SEQ ID NO: 1 is the nucleic acid sequence for exemplary PET tag5′-X₁X_(2(a))X₃X_(4(a))G_(x)-3′.

SEQ ID NO: 2 is the nucleic acid sequence for exemplary PET tag5′-X₁X_(2(a))X₃X_(4(a))G_(x)X_(5(n))-3′.

SEQ ID NO: 3 is the nucleic acid sequence for exemplary PET tag5′-X₁X_(2(a))X₃X_(4(a))-3′.

SEQ ID NO: 4 is the nucleic acid sequence for exemplary PET tag5′-TAMRA-AGGCGCATAGCGCCTGG-3′.

SEQ ID NO: 5 is the nucleic acid sequence for the C. parvum 18S ss rRNAsequence-specific reverse primer CryJVR 5′-ATTCCCCGTTACCCGTCA-3′.

SEQ ID NO: 6 is the nucleic acid sequence for the C. parvum 18S ss rRNAsequence-specific forward primer CryJVF 5′-GGTGACTCATAATAACTTTACGGAT-3′.

SEQ ID NOS: 7 and 8 are a forward and a reverse primer for TaqMan™amplification of the C. parvum 18S ss rRNA gene, respectively.

SEQ ID NO: 9 is a TaqMan™ probe for detection of the amplified C. parvum18S ss rRNA gene.

SEQ ID NOS: 10-27 are exemplary PET tags attached to a sequence-specificprimer for C. parvum 18S ss rRNA (ACTCATAATAACTTTACGGAT; nucleotides20-40 of SEQ ID NO: 10). One skilled in the art will appreciate the PETtag portion of SEQ ID NOS: 10-27 can be attached to othersequence-specific primers.

SEQ ID NOS: 28 and 29 are exemplary PET tag sequences.

SEQ ID NOS: 30-32 are PET primers that include a PET tag with zero, oneor two 3′-end G nucleotides, respectively, attached to asequence-specific primer for C. parvum 18S ss rRNA (ATGACGGGTAACGGGGAAT;SEQ ID NO: 7). One skilled in the art will appreciate that the PET tagportion can be attached to other sequence-specific primers.

SEQ ID NO: 33 is a reverse sequence-specific primer that can be used incombination with SEQ ID NOS: 30-32 to amplify C. parvum 18S ss rRNA.

SEQ ID NOS: 34-35 are PET forward and reverse primers, respectively,that include a sequence-specific primer for C. parvum 18S ss rRNA. Oneskilled in the art will appreciate the PET tag portion can be attachedto other sequence-specific primers.

SEQ ID NO: 36 is a Quas670 probe specific for C. parvum 18S ss rRNA.

SEQ ID NOS: 37-38 are PET forward and reverse primers, respectively,that include a sequence-specific primer for C. parvum 18S ss rRNA. Oneskilled in the art will appreciate the PET tag portion can be attachedto other sequence-specific primers.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS Abbreviations and Terms

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. The singular forms“a,” “an,” and “the” refer to one or more than one, unless the contextclearly dictates otherwise. For example, the term “comprising a nucleicacid molecule” includes single or plural nucleic acid molecules and isconsidered equivalent to the phrase “comprising at least one nucleicacid molecule.” The term “or” refers to a single element of statedalternative elements or a combination of two or more elements, unlessthe context clearly indicates otherwise. As used herein, “comprises”means “includes.” Thus, “comprising A or B,” means “including A, B, or Aand B,” without excluding additional elements.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting.

CT: crossing or cycle threshold)

PCR: polymerase chain reaction

PET: photoinduced electron transfer

3′-end: The end of a nucleic acid molecule that does not have anucleotide bound to it 3′ of the terminal residue. In some examples, aPET tag includes two or more G nucleotides at its 3′-end. In someexample, a PET tag is covalently linked or otherwise attached at its3′-end to the 5′-end of a sequence-specific primer directed to a targetnucleic acid.

5′-end: The end of a nucleic acid sequence where the 5′-position of theterminal residue is not bound by a nucleotide.

5′-end labeled nucleotide: The terminal residue at the 5′-end of anucleic acid molecule possessing a label (such as a label that iscovalently attached) capable of emitting a detectable signal. The labelcan be incorporated by enzymatic modification of the terminal nucleotideafter isolation of the nucleic acid molecule. In particular examples,the label can be a constituent moiety of a modified nucleotide substrateused in the synthesis of the nucleic acid molecule. In such examples,the label can be incorporated into the nucleotide at any position (suchas the α, β, or γ phosphate or the sugar) so long as it does notsignificantly interfere with polynucleotide synthesis.

Amplifying a nucleic acid molecule: To increase the number of copies ofa nucleic acid molecule. The resulting amplification products are called“amplicons.” In a particular example, a target nucleic acid molecule isamplified using the polymerase chain reaction (PCR) whereby a forwardprimer and a reverse primer are incubated with a target nucleic acidsequence under repeated cycles of DNA denaturation, annealing and primerextension. During primer extension, the primers are utilized by a DNApolymerase in the synthesis of a DNA strand complementary to the targetnucleic acid. Thus, each resulting DNA amplicon contains either anewly-extended forward primer or reverse primer. A primer extensioncycle is completed when the sample incubation conditions are changed todenature the newly synthesized dsDNA.

Complementary: Complementary binding occurs when a nucleotide forms ahydrogen bond to another nucleotide. In one example, the complementarynucleotides are present on a single nucleic acid molecule; for examplecausing this nucleic acid molecule to form a secondary structure such asa hairpin loop. In other examples, the complementary nucleotides arepresent on two different nucleic acid molecules, such as single-strandedDNA molecules, for example thereby forming a duplex (e.g.,double-stranded DNA). Normally, the base adenine (A) is complementary tothymidine (T) and uracil (U), while cytosine (C) is complementary toguanine (G). For example, the sequence 5′-ATCG-3′ of one portion of anucleic acid molecule can bond to 3′-TAGC-5′ of another portion of thesame nucleic acid molecule, for example to form a section of dsDNA. Inthis example, the sequence 5′-ATCG-3′ is the reverse complement of3′-TAGC-5′.

Nucleic acid molecules can be complementary to each other even withoutcomplete hydrogen-bonding of all bases of each molecule. For example,hybridization with a complementary nucleic acid sequence can occur underconditions of differing stringency in which a complement will bind atsome but not all nucleotide positions. In particular examples disclosedherein, the complementary sequences comprising a stem-loop structure aresufficiently complementary to maintain the stem structure even thoughone or more base pairs within the stem are non-complementary.

Denaturation: The conversion of one or more molecules from a folded to alinear physical conformation. Denaturation also refers to the separationof a partially or completely double-stranded nucleic acid molecule intoits single-stranded constituents. Molecular denaturation can occur uponchanges in temperature, salt concentration, or pH as described inSambrook et al. (Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y., 2001) and Ausubel et al. (In Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1998).

In particular examples, dsDNA is denatured into ssDNA during PCR byelevating the incubation temperature to 94° C. or greater for at leastone minute.

Detectable Signal: An indicator, such as a detectable physical quantityfrom which information can be obtained. In one example, a label emits asignal capable of detection, such as a fluorescent signal. When a labelis incorporated uniformly into a group of molecules, the presence of itsdetectable signal can be directly correlated with the number ofmolecules in a given sample. In some examples the detection of thesignal is dependant on the molecular context within which the signal isfound, such as its proximity to a molecular quencher. In other examples,such as particular fluorescent signals, the detection of the signalrequires external stimulus (for example, a particular wavelength oflight) for generation of the signal.

Fluorophore: A chemical compound, which when excited by exposure to aparticular wavelength of light, emits light (fluoresces), for example ata different wavelength of light. Exemplary fluorophores include, but arenot limited to: 6-carboxyfluorescein (6-FAM™ dye); 5-carboxyfluorescein(5-FAM™ dye); boron dipyrromethene difluoride (BODIPY® dye);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA™ dye); acridine,stilbene, 6-carboxy-fluorescein hexachloride (HEX™ dye), TET™ dye(Tetramethyl fluorescein), 6-carboxy-X-rhodamine (ROX™ dye), ALEXAFLUOR® 488, Texas Red® dye,2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE™ dye), Cy3®dye, Cy5® dye, VIC® dye (Applied Biosystems), LC Red 640, LC Red 705,Yakima yellow, as well as derivatives thereof. Any fluorophore can beused with the PET tags disclosed herein.

Also encompassed by the term “fluorophore” are luminescent molecules,which are chemical compounds which do not require exposure to aparticular wavelength of light to fluoresce; luminescent compoundsnaturally fluoresce. Therefore, the use of luminescent signals caneliminate the need for an external source of electromagnetic radiation,such as a laser.

A particular type of fluorophore is one whose fluorescence is quenchedin the presence of guanine (G), such as 6-FAM™ dye; 5-FAM™ dye; HEX™dye; ALEXA FLUOR® 488; boron dipyrromethene difluoride (BODIPY® dye); orN,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA™ dye). In one example,fluorescence is quenched in the presence of guanine by at least 25%,such as at least 50%, at least 75%, at least 80%, or at least 90%, ascompared to an amount of fluorescence in the absence of guanine (whereinboth are in the presence of the appropriate excitation wavelength oflight).

Hybridization: Hybridization of a nucleic acid occurs when twocomplementary nucleic acid molecules undergo an amount of hydrogenbonding to each other, or two different regions of a single nucleic acidmolecule undergo an amount of hydrogen bonding to one another. Thestringency of hybridization can vary according to the environmentalconditions surrounding the nucleic acids, the nature of thehybridization method, and the composition and length of the nucleicacids used. Calculations regarding hybridization conditions required forattaining particular degrees of stringency are discussed in Sambrook etal., Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2001); and Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes Part I, Chapter 2(Elsevier, New York, 1993). The T_(m) is the temperature at which 50% ofa given strand of nucleic acid is hybridized to its complementarystrand.

Increase in signal: To become greater in some way. A detectable increaseis one that can be detected, such as an increase in the intensity,frequency, or presence of an electromagnetic signal, such asfluorescence. In particular examples, the detectable increase can bedirectly correlated to the presence of a target nucleic acid moleculeand additionally to the quantity of a target nucleic acid molecule. Inother particular examples, differences in the increase of signal withina population of molecules are indicative of polymorphisms within thatpopulation.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule) has been substantially separated, produced apart from, orpurified away from other biological components such as cells. Nucleicacid molecules which have been “isolated” include nucleic acidsmolecules purified by standard purification methods, as well as thosechemically synthesized. Isolated does not require absolute purity, andcan include nucleic acid molecules that are at least 50% isolated, suchas at least 75%, at least 80%, at least 90%, at least 95%, at least 98%,at least 99% or even 100% isolated.

Label: An agent capable of detection, for example by spectrophotometry,flow cytometry, or microscopy. For example, a label can be attached to anucleotide, thereby permitting detection of the nucleotide, such asdetection of the nucleic acid molecule of which the nucleotide is a partof (e.g., a PET tag or PET primer). Examples of labels include, but arenot limited to, radioactive isotopes, enzyme substrates, co-factors,ligands, chemiluminescent agents, fluorophores, haptens, enzymes, andcombinations thereof. In some examples the label is one whose signal canbe quenched by two or more G nucleotides. Methods for labeling andguidance in the choice of labels appropriate for various purposes arediscussed for example in Sambrook et al. (Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y., 2001) and Ausubel et al.(In Current Protocols in Molecular Biology, John Wiley & Sons, New York,1998).

Ligase: An enzyme that can catalyze the joining of two molecules(“ligation”) by forming a new chemical bond. An exemplary ligase is DNAligase, which can link two nucleic acid molecules (e.g., a PET tag and asequence-specific primer) by forming a phosphodiester bond between thetwo molecules.

Nucleic acid molecule: A deoxyribonucleotide or ribonucleotide polymer,which can include analogues of natural nucleotides that hybridize tonucleic acid molecules in a manner similar to naturally occurringnucleotides. In a particular example, a nucleic acid molecule is asingle-stranded (ss) DNA or RNA molecule, such as a primer, cDNA,amplicon, or transcription product. In another particular example, anucleic acid molecule is a double-stranded (ds) molecule, such ascellular genomic DNA or viral genomic RNA.

Nucleotide: The fundamental unit of nucleic acid molecules. A nucleotideincludes a nitrogen-containing base attached to a pentose monosaccharidewith one, two, or three phosphate groups attached by ester linkages tothe saccharide moiety.

The major nucleotides of DNA are deoxyadenosine 5′-triphosphate (dATP orA), deoxyguanosine 5′-triphosphate (dGTP or G), deoxycytidine5′-triphosphate (dCTP or C) and deoxythymidine 5′-triphosphate (dTTP orT). The major nucleotides of RNA are adenosine 5′-triphosphate (ATP orA), guanosine 5′-triphosphate (GTP or G), cytidine 5′-triphosphate (CTPor C) and uridine 5′-triphosphate (UTP or U).

Nucleotides include those nucleotides containing modified bases,modified sugar moieties and modified phosphate backbones, for example asdescribed in U.S. Pat. No. 5,866,336 to Nazarenko et al. PET tags andsequence-specific primers can include one or more modified bases,modified sugar moieties or modified phosphate backbones.

Examples of modified base moieties which can be used to modifynucleotides at any position on its structure include, but are notlimited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N-6-sopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,methoxyarninomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid,pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-S-oxyacetic acid, 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine.

Examples of modified sugar moieties which can be used to modifynucleotides at any position on its structure include, but are notlimited to: arabinose, 2-fluoroarabinose, xylose, and hexose, or amodified component of the phosphate backbone, such as phosphorothioate,a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or aformacetal or analog thereof.

In particular examples, a nucleotide can be modified prior toincorporation into a growing nucleic acid chain so as to possess a labelcapable of emitting a detectable signal. Ideally, such modificationsallow for incorporation of the nucleotide into a growing nucleic acidchain. That is, they do not terminate nucleic acid synthesis. In otherparticular examples, a nucleotide is modified after synthesis of thenucleic acid molecule. An exemplary nucleotide modification is thecovalent attachment of a fluorophore.

Polymorphism: A variation in the nucleic acid sequence within apopulation of molecules. Polymorphisms may be differences in consecutiveor non-consecutive nucleotides within a particular sequence. Inparticular examples, a polymorphism is a difference in a single basepair. In other examples a polymorphism is 5, 10, 20, or greaterdifferences in nucleotide identity. In other examples a polymorphism maybe a deletion of sequence, an insertion of sequence, or an inversion ofsequence. Sequence differences in polymorphic nucleic acids will resultin differences in the rate and temperature at which the polymorphicmolecules will denature from dsDNA to ssDNA and anneal into dsDNA fromssDNA. In one example, a target sequence can contain one or morepolymorphisms, such as a polymorphism associated with disease.

Primer: A short nucleic acid molecule, such as an 8-nucleotide long DNAor RNA oligonucleotide. Longer primers can be about 10, 12, 15, 20, 25,30 or 50 nucleotides or more in length, such as 10-75, 10-50, 10-25,10-20, 10-15, 12-50 or 12-20 nucleotides. Primer extension occurs when aprimer is used to initiate the synthesis of a longer nucleic acidsequence. Primers can be annealed to a complementary target DNA strandby nucleic acid hybridization to form a hybrid between the primer andthe target DNA strand. The primer is then extended along the templatetarget DNA strand by a DNA polymerase enzyme. Forward and reverseprimers can be used for amplification of a nucleic acid sequence, forexample by PCR or other nucleic acid amplification methods.

Specificity of a primer for a target nucleic acid increases with thelength of complementary sequence possessed by the primer. Thus, forexample, a primer that includes 30 consecutive complementary nucleotideswill anneal to a target sequence with greater specificity than acorresponding primer of only 15 complementary nucleotides. Thus, toobtain greater specificity, probes and primers can be selected thatinclude at least 20, 25, 30, 35, 40, 45, 50 or more consecutivecomplementary nucleotides. Conversely, a PET tag described herein is aprimer that possesses little or no complementary sequence to a targetnucleic acid molecule, such that it is unable to hybridize to the targetmolecule under conditions of moderate or high stringency.

In particular examples, a 5′-end labeled PET tag can be covalentlyattached at its 3′-end to the 5′-end of a sequence-specific primer.

Photoinduced Electron Transfer (PET) primer: A PET tag covalentlyattached at its 3′-end to a sequence-specific primer, such that underconditions suitable for nucleic acid amplification, the hairpin isdenatured, moving the 5′-end label out of proximity from the at leasttwo adjacent G residues, allowing the 5′-end label to emit a detectablesignal.

Photoinduced Electron Transfer (PET) tag: A short nucleic acid moleculecontaining a stem-loop structure, wherein the stem-loop structurepositions a 5′-end label into proximity with at least two adjacent Gresidues (e.g., at the 3′-end of the PET tag) such that the G residuesquench a detectable signal from the 5′-end label. In particular examplesa PET tag is at least 10 nucleotides, at least 12 nucleotides, such as10-20 nucleotides, for example 12, 13, 14 or 15 nucleotides.

Proximity: A measure of nearness, for example when a detectable signalfrom a label is quenched if the label is in sufficient proximity to thequencher of that label. In particular examples, the detectable signalfrom the 5′-end label of a PET tag is significantly quenched when placedinto proximity with at least two adjacent G residues.

Quantifying a nucleic acid molecule: Determining or measuring a quantity(such as a relative quantity) of a nucleic acid molecule present, suchas the number of amplicons or the number of nucleic acid moleculespresent in a sample. In particular examples, it is determining therelative amount or actual number of nucleic acid molecules present in asample.

Quencher: A molecular species that can reduce a detectable signal from alabel. In particular examples, a quencher can be at least twoconsecutive G residues that quench the signal from a label at the 5′-endof a PET primer or PET tag.

Quenching a signal: A reduction of detectable signal from a label, suchas a reduction in fluorescence emission. For example, quenching of adetectable fluorescent signal emitted from a label at the 5′-end-labelednucleotide on a PET tag occurs when the label, through sequence-directedsecondary structure, is placed in sufficient proximity to a quencher(such as at least two consecutive G residues) that the quencher reducesthe detectable signal from the label on the 5′-end labeled nucleotide.

Real-time quantitative PCR: A method for detecting and measuringproducts generated during each cycle of a PCR, which are proportionateto the amount of template nucleic acid prior to the start of PCR. Theinformation obtained, such as an amplification curve, can be used toquantitate the initial amounts of template nucleic acid sequence.

Sample: Biological specimens such as samples containing biomolecules,for example nucleic acid molecules (e.g., genomic DNA, cDNA, RNA, ormRNA). Exemplary samples are those containing cells or cell lysates froma subject, such as those present in peripheral blood (or a fractionthereof such as serum), urine, saliva, tissue biopsy, cheek swabs,surgical specimen, fine needle aspirates, amniocentesis samples andautopsy material.

Sequence-specific primer: A short nucleic acid molecule possessingsequence that can substantially hybridize with a target nucleic acidmolecule under moderately stringent or highly stringent conditions. Inparticular examples, a sequence-specific primer is covalently attachedat its 5′-end to the 3′-end of a PET tag can be used to detect thepresence of a target nucleic acid molecule. In other examples, asequence-specific primer is used for location-specific amplification ofa target nucleic acid molecule using PCR. In some examples, asequence-specific primer is at least 8 nucleotides, such as at least 10,at least 15, at least 20 nucleotides, for example 8-50, 8-25, 8-20,8-15, 10-20, or 12-20 nucleotides.

Signal: An indicator, such as a detectable physical quantity from whichinformation can be obtained. In one example, a label emits a signalcapable of detection, such as a fluorescent signal.

Stem-loop: As shown in FIGS. 1A and 2, a molecular secondary structurewherein two portions of a linear molecule (e.g., 20 of FIG. 2) possesssufficient affinity (e.g., complementarity) to fold into adouble-stranded stem (e.g., 18 of FIGS. 1A and 1B) that is connected bya single-stranded loop (e.g., 22 of FIGS. 1A and 1B). A nucleic acidstem-loop is the result of two inverted repeat sequences connected bythree or more nucleotides. In particular examples, the inverted repeatsare less than 100% complementary, but the overall sequence issufficiently complementary to maintain the stem structure.

Target nucleic acid sequence or molecule: A pre-selected nucleic acidmolecule, for example whose detection or sequence is desired. The targetnucleic acid molecule need not be in a purified form. Various otherbiomolecules can also be present with the target nucleic acid molecule.For example, the target nucleic acid molecule can be present in a cellor a biological sample (which can include other nucleic acid moleculesand proteins).

Under conditions sufficient for: A phrase that is used to describe anyenvironment that permits the desired activity. An example includesincubating forward and reverse primers with a sample under conditionssufficient to allow amplification of a target nucleic acid molecule inthe sample. Another particular example includes conditions sufficientfor determining whether the target nucleic acid molecule is present in asample, such as a target nucleic acid molecule containing one or morepolymorphisms.

Photoinduced Electron Transfer (PET) Tags and Primers and Methods ofMaking

Disclosed herein are photoinduced electron transfer (PET) nucleic acidmolecules (referred to herein as PET tags and PET primers) that can beused in nucleic acid amplification to detect the presence of a targetnucleic acid molecule. The PET tag sequence is generic and withoutsignificant specificity for any particular nucleic acid sequence. Forexample, rather than hybridize specifically to a target nucleic acid,PET tags can be ligated or synthesized at their 3′-end to a forwardand/or reverse amplification primer that contains significant sequencespecificity for the target nucleic acid molecule. The resulting nucleicacid (referred to here as a PET primer) can be used to detect a targetnucleic acid molecule.

Upon incorporation of a PET primer (e.g., one that is covalentlyattached to a target-specific sequence) into a newly-synthesizedamplicon, a quenched detectable signal from a label at the PET primer5′-end is moved away from quenching nucleotides contained therein. Thesignal is thus de-quenched and detectable, and indicates the presence ofthe target nucleic acid. In some examples, the signal can be detectedafter each amplification cycle to quantitate the amount of amplifiedtarget nucleic acid in real time, as in real-time PCR or real timeRT-PCR. In other examples, the signal can be detected afteramplification is completed. In other particular examples the signal fromthe incorporated PET primer can be used to detect the presence ofnucleotide polymorphisms, for example by monitoring the signal duringamplicon denaturation by methods well known to the art. Althoughparticular PET tag sequences are provided herein (e.g., see Table 2),the disclosure is not limited to these specific examples.

FIGS. 1A-B and 2 show an exemplary PET nucleic acid molecule tag whennot hybridized to its complementary sequence (e.g., stem-loop structurepresent), and when hybridized to its complementary sequence (e.g.,stem-loop structure absent) as part of an amplicon, respectively. ThePET tag 10 includes a 5′-end nucleotide 12 with a label 14 capable ofemitting a detectable signal. The 5′-end label 14 is located at the baseof a stem-loop, the stem 18 of which is formed by two inverted repeats(20, FIG. 2) of sufficient length and complementary nucleotide sequenceto anneal one to another. Each inverted repeat 20 is separated by threeor more non-complementary nucleotides to form the loop 22 of thestem-loop. The structure of the stem-loop is such that the 5′-end label14 is positioned into proximity with at least two consecutive Gnucleotides (or isoC or isoG) 24 located at the 3′-end of the stem-loopstructure. The proximity of the G nucleotides 24 to the 5′-end label 14quenches the detectable signal from the label 14. The 3′-end portion ofthe PET tag 10 follows the at least two consecutive G nucleotides 24. Insome examples, the PET tag includes one or more nucleotides 26 after theat least two consecutive G nucleotides 24, such as 1-50 nucleotides,such as 1, 2, 3, 4, 5, or 10 nucleotides.

The 5′-end nucleotide 12 can be any nucleotide that can be covalentlymodified to contain a label with a detectable signal. In particularexamples, the 5′-end nucleotide 12 is a T, A, G, or C nucleotide. Inother particular examples, the 5′-end nucleotide 12 is a T, A, or Cnucleotide. In other particular examples, the 5′-end nucleotide 12 isany nucleotide except G. In particular examples, the 5′-end nucleotide12 is any nucleotide analog that contains a label 14 that emits adetectable signal.

The 5′-end label 14 can be any label that is capable of emitting adetectable signal. In particular examples, the 5′-end label 14 is afluorophore. In one example, the fluorophore emits a fluorescent signalthat is quenched when the label is brought into proximity of the atleast two consecutive G nucleotides 24. The signal can be decreased byany detectable amount, such as at least 10%, at least 30%, at least 50%,at least 70%, at least 90%, or even 100%. Particular examples offluorophores that can be used include, but are not limited to,6-carboxyfluorescein (6-FAM™ dye); 5-carboxyfluorescein (5-FAM™ dye);boron dipyrromethene difluoride (BODIPY® dye);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA™ dye); ALEXA FLUOR® 488;acridine; stilbene; 6-carboxyfluorescein hexachloride (HEX™ dye); TET™dye; ROX™ dye; Texas Red® dye; JOE™ dye; Cy3® dye; Cy5® dye; VIC® dye;LC Red 640; LC Red 705; Yakima yellow; as well as derivatives thereof.In another example, the label is not a quencher.

The 5′-end label 14 can be covalently attached to the PET tag 10 at anyavailable moiety of the 5′-end nucleotide 12. In particular examples,the 5′-end label 14 is covalently attached at the triphosphate of the5′-end nucleotide 12. In other particular examples, the 5′-end label 14is covalently attached at any available moiety of the nitrogenous baseof the 5′-end nucleotide 12. In other particular examples, the 5′-endlabel 14 is covalently attached to any available moiety of the sugarcomponent of the 5′-end nucleotide 12. Covalent attachment of the 5′-endlabel 14 to the triphosphate, nitrogenous base, or sugar of the 5′-endnucleotide 12 can be accomplished according to standard methodology wellknown in the art as discussed, for example in Sambrook et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 2001) andAusubel et al. (In Current Protocols in Molecular Biology, John Wiley &Sons, New York, 1998).

The 5′-end nucleotide 12 is located at the base of a stem-loop structure16. The stem-loop 16 (linear in FIG. 2) functions to bring the 5′-endlabel 14 within proximity of the at least two consecutive G nucleotides24, which quench the signal from the label 14. The stem 18 of the stemloop structure 16 is composed of two lengths of nucleotide sequence 20(FIG. 2) that are of sufficient complementarity one to another to stablybase pair. In particular examples, the stem 18 can be composed of twoinverted repeats 20 of 100% complementarity to each other. In otherparticular examples, the stem 18 is composed of sequences 20 that are atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, or atleast 95% complementary to each other. The length of the stem 18 can beany number of nucleotides, so long as the stem can be stably maintainedunder non-denaturing conditions. In particular examples each of thecomponent sequences portions 20 of the stem 18 is at least 3, at least 4at least 5, at least 6, at least 7, at least 8, or at least 9nucleotides, such as 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In otherparticular examples each of the two component sequences 20 of the stem18 is 10 or more nucleotides. In some examples, each of the twocomponent sequences 20 of the stem 18 is 3-5 or 3-10 nucleotides.

The loop 22 of the stem-loop structure 16 connects the two componentsequences 20 of the stem. The loop 22 can be composed of any number ofnucleotides and any sequence such that the stem-loop structure 16 ismaintained in order to quench the detectable signal from the 5′-endlabel 14 as described herein. In particular examples, the loop 22 is 3,4, 5, or 6 nucleotides. In other particular examples, the loop 22 is atleast 7 nucleotides, such as 7-12 nucleotides. In other particularexamples, the loop 22 is at least 10 nucleotides. In particularexamples, the loop 22 can be any trinucleotide sequence. In otherparticular examples, the loop 22 does not contain C or G nucleotides. Inother particular examples, the loop 22 can be any trinucleotide sequencethat does not contain C or G nucleotides. In other particular examples,the loop 22 is TAA, ATA, AAT, TTA, TAT, ATT, TTT, or AAA.

In particular examples, the PET tag 10 includes at least two consecutiveG nucleotides 24 at the 3′-end of the stem-loop structure 16. However,in some examples the at least two consecutive G nucleotides are insteadpresent at the 5′-end of a sequence-specific primer attached to the3′-end of the PET tag. One skilled in the art will appreciate that isoCor isoG can be used alternatively or in addition to G. The G nucleotides24 quench the detectable signal from the 5′-end label 14 when the 5′-endlabel 14 is brought into proximity with the consecutive G nucleotides 24by the stem-loop structure 20 (see FIGS. 1A and B). However, when thePET tag is hybridized to its complementary sequence (for example whenincorporated into an amplicon), the stem-loop structure 16 linearizesmoving the G nucleotides 24 away from proximity to label 14, and thusthe G nucleotides 24 cannot significantly quench the detectable signalfrom the 5′-end label 14 and the detectable signal from the label 14 isemitted and can be detected (see FIG. 2). In particular examples, theconsecutive G nucleotides 24 can include 2, 3, 4, 5, or 6 consecutive Gnucleotides. In other particular examples the consecutive G nucleotides24 include at least 7 consecutive G nucleotides. As shown in FIG. 2, ina particular example, the PET tag stem-loop structure 16 is linearizedas a result of its incorporation into a nucleic acid amplicon.

As shown in FIG. 1B, the PET tag in some examples includes additionalnucleotides 26 at the 3′-end of the PET tag following the at least twoconsecutive G nucleotides 24. For example, the additional nucleotides 26can be composed of 0, 1, 2, 3, 4, 5, 8, 10, 15, 20 or more nucleotides,such as at least 8 nucleotides.

In specific embodiments, the PET tags 10 disclosed herein can be atleast 12 nucleotides in length, such as 12 to 20 nucleotides, forexample 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. Inother specific examples, the PET tags can be between 22 and 30nucleotides long, such as 22, 23, 24, 25, 26, 27, 28, 29, or 30nucleotides. In other specific examples the PET tags 10 can be 35, 40,45, or 50 nucleotides long.

As shown in FIGS. 3A and 3B, a PET tag 10 can be linked (e.g., ligated,synthesized, or attached) at its 3′-end to the 5′-end of asequence-specific primer sequence 30, thereby generating a labeledsequence-specific primer sequence 32 (also referred to herein as a PETprimer). Such PET primers can be generated using routine methods, suchas by synthesizing a nucleic acid molecule that includes a PET tag and asequence-specific primer, or by ligating a PET tag to asequence-specific primer. In some examples, the target-specific primer30 is added to the PET tag 10 via the at least two consecutive Gnucleotides 24 (FIG. 3A). In other examples, the target-specific primer30 is added to the PET tag 10 via additional nucleotides 26 (FIG. 3B).The labeled sequence-specific primer 32 (which in some examples isisolated) can then be used in an amplification reaction, such as a PCRor an RT-PCR reaction. The sequence-specific primer 30 can recognize atarget nucleic acid of interest, such as a pathogen nucleic acidsequence, for example a viral, fungal, bacterial, or parasitic DNA orRNA sequence. In another example, a target nucleic acid sequence, suchas a DNA or RNA sequence, is a nucleic acid sequence whose expression isaltered in response to a disease, such as cancer. In some examples, thetarget nucleic acid sequence is one whose gene expression is to bedetermined. The sequence-specific primer 30 can be any length thatpermits amplification of the desired nucleic acid molecule. Inparticular examples, a sequence-specific primer 30 is at least sixnucleotides, such as at least 9, at least 10, at least 12, at least 15,at least 20, at least 25, at least 30, at least 35, at least 40, or atleast 50 nucleotides. In particular examples the sequence-specificprimer is between 6 and 100, 9 and 50, or 9 and 20 nucleotides.

In particular examples the PET tag includes the sequence5′-X₁X_(2(a))X₃X_(4(a))G_(x)-3′ (SEQ ID NO: 1), wherein X₁ is the 5′-endnucleotide of the PET tag and includes a detectable label (12, FIGS. 1Aand B and FIG. 2), wherein X₂ and X₄ (20, FIG. 2) include the nucleotidesequences of length a of sufficient complementarity to form the stem ofthe stem-loop structure, wherein X₃ (22, FIGS. 1A and B and FIG. 2)includes the loop of the stem-loop structure, wherein G_(x) (24, FIGS.1A and B and FIG. 2) includes the at least two consecutive G nucleotidessuch as 2, 3, 4, 5, or 6 nucleotides. In another example, a PET tagincludes the sequence 5′-X₁X_(2(a))X₃X_(4(a))G_(x)X_(5(n))-3′ (SEQ IDNO: 2), wherein X₅ (26, FIGS. 1B and 3B) is 0 or more nucleotides suchas 0, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, or 35 nucleotides. In otherembodiments, X₅ is 1 or more nucleotides. In another example, a PET tagincludes the sequence 5′-X₁X_(2(a))X₃X_(4(a))-3′ (SEQ ID NO: 3), whereinG_(x) of the PET tag (24, FIGS. 3A and B) is the 5′-nucleotides of thesequence specific primer 30 instead part of the PET tag attached to thesequence specific primer 30.

In particular examples, X₁ is any nucleotide, such as A, C, T, or G orany modification or nucleotide analog known to a person skilled in theart. In other examples, X₁ is any nucleotide except for G, such as A, C,T, or any modification or nucleotide analog thereof known to a personskilled in the art. In other examples, X₁ is A, C, or T. In particularexamples, a is 3 or more nucleotides such as 3, 4, 5, 6, 7, 8, 9, or 10nucleotides. In other specific examples, the sequence defined byX_(2(a)) and X_(4(a)) possess at least 50% complementarity to oneanother such as at least 50%, 60%, 70%, 80%, or 90% or 95%complementarity. In other specific examples, the sequences defined byX_(2(a)) and X_(4(a)) are 100% complementary to one another. Inparticular examples, X₃ is at least 3 nucleotides such as 3, 4, 5, 6, 7,8, 9, or 10 nucleotides. In other particular examples, the nucleotidesrepresented by X₃ do not include C or G. In other particular examples X₃is a trinucleotide sequence, for example TAA, ATA, AAT, TTA, TAT, ATT,TTT or AAA.

In a specific example, the PET tag is 5′-TAMRA-AGGCGCATAGCGCCTGG-3′ (SEQID NO: 4). One skilled in the art will appreciate that TAMRA can bereplaced with another fluorophore. Other exemplary PET tags are providedin the examples below.

Kits

The present disclosure provides kits that include one or more PET tags,such as a PET tag ligated or otherwise attached to a sequence-specificprimer. For example, the kit can include one or more (such as two ormore, for example, 2, 3, 4, 5, or 6) labeled sequence-specific primersthat include a PET tag generated using the methods provided herein.

In some examples, the kits further include ligase, for example to permitligation of a PET tag to the 5′ end of a forward or a reverse targetsequence-specific primer, thereby generating a PET primer.

In some examples, the kits include one or more forward or reverse targetsequence-specific primers, such as forward and reverse primers thatrecognize a specific pathogen or a specific nucleic acid sequence whoseexpression is changed in response to a disorder. For example, the kitcan include forward and reverse primers that can be used to amplify thenucleic acid sequence of a particular pathogen, such as a viral,bacterial, parasitic, or fungal target nucleic acid sequence. In oneexample, the forward and reverse primers can be used to amplify aparticular human nucleic acid sequence, such target nucleic acidsequences can be associated with a disease, such as cancer. In someexamples, the PET tag in the kit is already attached to the 5′-end ofthe forward or reverse primer. In other examples, the PET tag in the kitis separate from the forward or reverse primer, and can be ligated tothe forward or reverse primer by a user.

Kits can also include other reagents, such as those used for PCRamplification. Examples include buffers, dNTPs, polymerase, andcombinations thereof. In one example, kits include reagents fordetection of a label on the PET tag, such as a chemiluminescentdetection reagent.

The components of the kit can be present in separate, labeledcontainers.

Methods of Nucleic Acid Detection

The disclosed PET nucleic acid molecules and labeled sequence-specificprimers can be used in any nucleic acid amplification reaction todetermine whether a particular target nucleic acid sequence is present,such as a DNA or RNA molecule. For example, methods are disclosed fordetecting a target nucleic acid molecule. In particular examples, themethod includes incubating a sample containing nucleic acids with a PETtag attached to a forward or a reverse target sequence-specific primer(referred to herein as a PET primer), and with a corresponding forwardor reverse target sequence-specific primer which does not contain thePET tag. In other examples, both the forward and the reverse targetsequence contain a PET tag. The PET tag associated with thesequence-specific forward and reverse primer may be the same ordifferent. In some examples, the sequences of the PET tag associatedwith the sequence-specific forward and reverse primer is the same, butthe label on each is different. As described above, the 3′-end of thePET tag can be ligated to the 5′-end of the forward or reversesequence-specific primer (e.g., see FIGS. 3A and B).

In a particular example, the sample, a forward primer containing a PETtag, and a reverse primer not containing a PET tag, or a forward primernot containing a PET tag and a reverse primer containing a PET tag, areincubated under conditions sufficient to permit amplification of thetarget nucleic acid. For example, the reaction can include dNTPs,polymerase, and MgCl₂.

Any primer extension amplification method can be used, and such methodsare well known in the art. Particular examples include, but are notlimited to: real-time PCR (for example see Mackay, Clin. Microbiol.Infect. 10(3):190-212, 2004), Strand Displacement Amplification (SDA)(for example see Jolley and Nasir, Comb. Chem. High Throughput Screen.6(3):235-44, 2003), self-sustained sequence replication reaction (3SR)(for example see Mueller et al., Histochem. Cell. Biol. 108(4-5):431-7,1997), ligase chain reaction (LCR) (for example see Laffler et al., Ann.Biol. Clin. (Paris). 51(9): 821-6, 1993), or transcription mediatedamplification (TMA) (for example see Prince et al., J. Viral Hepat.11(3):236-42, 2004),

An increase in detectable signal from the label on the labeled PETprimer is monitored, wherein a significant increase in signal indicatesthe presence of the target nucleic acid sequence, and wherein nosignificant increase in signal indicates that the target nucleic acidmolecule is not present in the sample. The increase in detectable signalcan be monitored by any instrument that can detect the detectablesignal. In particular examples, the instrument that can detect thedetectable signal can be a spectrophotometer. In other particularexamples, the instrument that can detect the detectable signal can be areal-time PCR thermocycler. The increase in signal can be compared to acontrol, such as a signal present at an earlier time-point, such asprior to nucleic acid amplification. In some examples, the increase isrelative to a negative control, such as a sample known not to containthe target DNA or a sample incubated with primers that are unlabeled. Insome examples, the increase is relative to a known value or range ofvalues expected in the absence of the target sequence. In comparison tothe control signal, the increase can be at least 10%, at least 20%, atleast 30%, at least 40%, at least 50% at least 60%, at least 70%, atleast 80%, at least 90%, at least 100%, at least 200% at least 1000% orgreater increase. The detectable signal increases in a predictablemanner that permits determination of whether or not a target nucleicacid sequence is present in a sample. In some examples, the increase indetectable signal allows for quantification of an amount of targetnucleic acid sequence present in a sample.

For example, when the label is a fluorophore that can be quenched in apredictable manner by being in proximity to the at least two consecutiveG nucleotides at the 3′-end of the PET tag (or the first two5′-nucleotides of the sequence-specific primer), an increase influorescent signal during nucleic acid amplification indicates thepresence of the target nucleic acid sequence in the sample, while nosignificant increase in fluorescent signal during nucleic acidamplification indicates that the target nucleic acid sequence is notpresent in the sample.

In some examples, the increase in signal is monitored during theamplification reaction, for example in real time as the amplicons areformed. For example, the detectable signal from the 5′-end label presenton the PET tag is quenched when the amplification primers are freelyfloating in the nucleic acid amplification reaction mixture. Duringnucleic acid amplification, when the polymerase synthesizes nucleic acidamplicons, the primer, including the labeled PET tag, is incorporatedinto the amplicon and the stem-loop of the PET tag is denatured,removing the 5′-end label from proximity with the at least twoconsecutive G nucleotides (that is, the distance between label and Gnucleotides is increased). Thus, the signal from the label will increaseas it becomes incorporated into the double-stranded amplicon molecule.As more amplicons are produced during nucleic acid amplification, thesignal of the reaction mixture will increase. The increase in signal canbe monitored using any commercially available system. This increase insignal permits detection of a target nucleic acid sequence in thereaction.

In one example where the label is a fluorophore, the increase in signalmonitored during the amplification reaction is an increase influorescence. The fluorescence of the fluorophore is quenched when theprimers are freely floating in the nucleic acid amplification reactionmixture. During nucleic acid amplification, when polymerase synthesizesnucleic acid amplicons, the primer, including the labeled PET tag, isincorporated into the amplicon. The fluorescence of the incorporatedprimer increases several-fold due to dequenching of the detectablesignal by its incorporation into the double-stranded amplicon moleculeand movement out of proximity with the at least two consecutive Gs inthe PET tag. As more amplicons are produced during nucleic acidamplification, the overall fluorescence of the reaction mixtureincreases. The increase in fluorescence can be measured and observed,for example by using a commercially available nucleic acid amplificationsystem capable of measuring fluorescence (such as real-time PCRthermocyclers). An increase in fluorescent signal indicates the presenceof a target nucleic acid sequence in the reaction.

Target nucleic acid molecules can be detected after nucleic acidamplification. For example, the methods can include incubating a samplecontaining or thought to contain the target nucleic acid molecule with aforward primer and a reverse primer that are specific for the targetnucleic acid molecule. Either the forward primer or the reverse primeris linked at its 5′-end to the 3′-end of a PET tag under conditionssufficient to allow amplification of the target nucleic acid molecule(such as real-time PCR conditions). However, in some examples, both theforward and the reverse primer are linked at their 5′-ends to the 3′-endof a PET tag. The amplification results in the generation of labeledamplicons. Each amplicon is exposed to conditions that permitdenaturation of the amplicon into single-stranded nucleic acidmolecules, and then exposed to conditions that permit rehybridization ofthe strands. During each cycle of amplicon synthesis, the resulting PETprimer is incorporated into a double-stranded nucleic acid molecule,denaturing the stem-loop structure of the PET tag. This results in anincrease in detectable signal, for example relative to the detectablesignal from the label before the formation of double-stranded DNA. Anincrease in signal indicates that the target nucleic acid molecule ispresent in the sample, and no significant change in signal indicatesthat the target nucleic acid molecule is not present in the sample.

In particular examples, PET primers can be used to detect multipletarget nucleic acids, for example in a single reaction. In suchexamples, a plurality of PET tags can be ligated to the 5′-end of aplurality of target-specific forward and/or reverse primers. The 5′-endlabels in such examples can be fluorescent labels that each emit afluorescent signal at different wavelengths such that the presence of aplurality of target nucleic acids can be detected. For example, fortarget sequence 1, a PET-forward primer can be labeled with HEX, and fortarget sequence 2 a PET-forward primer can be labeled with 6-FAM, suchthat increase in HEX indicates the presence of target sequence 1, whilean increase in 6-FAM signal indicates the presence of target sequence 2.In particular examples, the presence of multiple target nucleic acidscan be monitored in real time as in real-time PCR, for example in one ormore amplification reactions.

In addition to determining whether a particular target nucleic acidmolecule is present, the method can further include quantifying thetarget nucleic acid molecule. In one example quantification includescomparing a signal to a reference value. Exemplary reference valuesinclude an expected amount of signal from a known amount of nucleicacid.

In other or additional examples, the change (e.g., increase or decrease)in signal is monitored after the amplification, for example by exposingthe resulting amplicons to a melting procedure to denature thedouble-stranded amplicons. During the denaturation, a change in signalis detected. The resulting signals, such as decreasing fluorescence (seeFIG. 8), can indicate polymorphisms in the nucleic acid amplicons.Therefore, melting curve analysis can be used to confirm the presence ofa target nucleic acid sequence, and can also be used to distinguishpolymorphisms in amplicons.

Samples containing nucleic acid molecules can be obtained from anyappropriate specimen, for instance blood or blood-fractions (such asserum). Techniques for acquisition of such samples are well known in theart (for example see Schluger et al. J. Exp. Med. 176:1327-33, 1992, forthe collection of serum samples). Serum or other blood fractions can beprepared in the conventional manner. For example, about 200 μL of serumcan be used for the extraction of DNA for use in amplificationreactions. In some examples, RNA is extracted and used in anamplification reaction (such as reverse-transcriptase PCR). Commerciallyavailable kits can also be used to obtain nucleic acid molecules from abiological sample prior to amplification.

Once a sample has been obtained, the sample can be used directly,concentrated (for example by centrifugation or filtration), purified, orcombinations thereof. In one example, DNA is prepared from the sample,yielding a nucleotide preparation that is accessible to, and amenableto, nucleic acid amplification. Similarly, RNA can be prepared using acommercially available kit (such as the RNeasy Mini Kit, Qiagen,Valencia, Calif.).

EXAMPLE 1 Comparison of PET Tag to TaqMan® Primers

This example describes methods used to compare the TaqMan® assay to themethod of the present disclosure which uses the disclosed PET tags andprimers. Cryptosporidium parvum was used as a model system; however oneskilled in the art will appreciate that similar methods can be used toamplify any target nucleic acid molecule of interest using the disclosedPET nucleic acid molecules.

The primers were prepared as follows. Oligonucleotide primers weresynthesized on automated DNA synthesizers (Applied Biosystems, FosterCity Calif.) utilizing standard phosphoramidite chemistry. The PET tagof SEQ ID NO: 4 does not show any homology to Cryptosporidium spp.sequences. TAMRA (NNN′N′-tetramethyl-6-carboxyrhodamine) was added tothe 5′-end of the oligo during synthesis using a C6-TAMRA-dTphosphoramidite (Glen Research, Sterling Va.) to produce the end labeledprimer of sequence 5′TAMRA-AGGCGCATAGCGCCTGG 3′ (SEQ ID NO: 4). Fortarget specific nucleic acid amplification and detection, the TAMRA-endlabeled PET tag was ligated to the 5′-end of the sequence-specificreverse primer CryJVR: 5′-ATTCCCCGTTACCCGTCA-3′ (SEQ ID NO: 5) toproduce PET-CryJVR. Also used in nucleic acid amplification was forwardprimer CryJVF: 5′-GGTGACTCATAATAACTTTACGGAT-3′ (SEQ ID NO: 6). Bothforward and reverse sequence-specific primers correspond to C. parvum18S ss rRNA sequence (GenBank Accession # AY458612).

A stock of C. parvum oocysts contained 6×10⁸ oocysts/mL. The titers ofC. parvum oocyts stocks were determined based on hemocytometermicroscopy counts. DNA was extracted using a standard nucleic acidextraction method and the resulting DNA was serially diluted and storedat −70° C. until use. Standard curves were generated using 10³ to 10⁻²oocysts. For generation of standard curves, the crossing threshold (CT)(i.e., cycle threshold) values were plotted (y-axis) against thelogarithm of the input copy numbers (x-axis). Appropriate negativecontrols were included in each run. To assess the log-linearrelationship of the assays, the linear regression and regressioncoefficients (R²) were calculated. The oocyst numbers do not correspondto the exact number of RNA molecules for 18S, since each oocyst contains20 copies of 18S ssrRNA gene.

Real-time PCR amplification was carried out using the iCycler iQ4®platform (Bio-Rad, California, USA) platform. The reaction mixturecontained primers at concentrations of 250 nM of each forward andreverse primer, 2 μl of DNA, 10 μl of 2× QuantiTect® Probe PCR kitMaster Mix (Qiagen, Valencia, Calif.), and nuclease-free water to afinal volume of 20 μl. The amplification reaction consisted of a hotstart step at 95° C. for 15 minutes to activate the HotStarTaq® DNApolymerase. This was followed by forty five cycles of amplificationincluding denaturation at 95° C. for 10 seconds and annealing/extensionat 60° C. for 40 seconds. Fluorescence signals were collected at the endof the annealing step in channel 2 (Excitation 555 nm/Emission 576 nm).

For the TaqMan® assay, the primers and probe used are listed in Table 1.The TaqMan® probe was labeled with FAM (6-Carboxy-fluorescein) at the5′-end and with Black Hole Quencher® dye at the 3′-end (CDCBiotechnology Core Facility, Atlanta, Ga.). Amplifications were carriedout using the iCycler iQ4® platform (Bio-Rad, California, USA) for atotal of 45 cycles. For TaqMan® PCR, the 20 μl reaction contained 10 μlof 2× QuantiTect® Probe PCR kit Master Mix (Qiagen, Valencia, Calif.), 2μl of DNA, and primers and probe at concentrations of 250 and 100 nMrespectively. Prior to amplification, denaturation was carried out at95° C. for 15 minutes, followed by 45 PCR cycles at 95° C. (10 seconds)and annealing/extension at 60° C. for 40 seconds. Fluorescence signalswere collected at the end of the annealing step in channel 1 (490 nm).

TABLE 1 Sequences used in the TaqMan ®  real-time PCR assayPrimer or probe Sequence (5′-3′) Position* SEQ ID No. 18S ssrRNA*JVAF (forward) ATGACGGGTAACGGGGAAT 100-118 7 JVAR (Reverse)CCAATTACAAAACCAAAAAGTCC 258-236 8 JVAP (Probe)FAM-CGCGCCTGCTGCCTTCCTTAGATG-BHQ 161-185 9 *Position based on GenBankaccession #AY458612 for 18S small subunit ribosomal RNA gene.

Slopes, regression coefficients, and PCR amplification efficiency curvesfor both PET primer and TaqMan® probe assays were calculated usingiCycler iQ® software; efficiency (E) was calculated according to theequation E=10^((−1/slope)).

As shown in FIG. 4, nucleic acid amplification with the disclosed PETPCR assay demonstrates a dynamic range of detection from 6000 oocysts to0.6 oocysts per PCR reaction. As shown in FIG. 5, the logarithmic plotof this data presents the relationship between the concentration of DNAand CT values. As shown in FIG. 6, the TaqMan® assay exhibits similarsensitivity as the PET primer assay in nucleic acid amplification for adynamic range of detection from 6000 oocysts to 0.6 oocysts per PCRreaction. Likewise, in FIG. 7 the logarithmic plot of this data presentsa similar relationship between the concentration of DNA and CT values incomparison to the PET PCR assay. In both detection methods the samelevel of sensitivity was achieved. A seed level of 0.06 oocysts was notdetected by either method.

EXAMPLE 2 Melting Curve Analysis to Detect Polymorphisms

This example describes methods used to detect polymorphisms using thedisclosed PET tags. Similar methods can be used to detect any targetnucleic acid molecule of interest using the disclosed PET nucleic acidmolecules.

Melting curve analysis of PET primer assay products was performed afteramplification (as described in Example 1), and consisted of 1 minute at95° C., followed by 1 minute at 55° C., and 80 10 second steps with a0.5° C. increase in temperature at each step. Threshold values forthreshold cycle determination were generated automatically by theiCycler iQ® software.

Lack of variation in PCR products and the absence of primer dimers wereascertained from the melt curve profile of the PCR products. The meltingtemperature (Tm) for each sample was used to verify the specificity ofthe real-time plot. As shown in FIG. 8, the melting curve analysis forthe PET PCR assay of the 18S ssrRNA gene target at differentconcentrations (as in Example 1) confirms the specificity of the PETprimers.

EXAMPLE 3 Exemplary PET Tags

This example provides an additional 18 exemplary PET tags. Although theprimers shown in Table 2 include target-specific sequences for the C.parvum 18S ssrRNA gene, one skilled in the art will appreciate that thetarget-specific portion of SEQ ID NOS: 10-27 (underlined portion inTable 2), can be replaced with other desired target-specific sequences.That is, the PET tags in Table 2 (non-underlined portion) can be usedwith other desired target-specific sequence primers.

Table 2 shows labeled target-specific sequences that include a PET tagportion (not underlined) and a target specific portion (underlined).These primers include different numbers of nucleotides in the loop(e.g., 16 of FIG. 2) and different numbers of consecutive Gs (e.g., 24of FIG. 2). These primers were evaluated for their ability to amplify aC. parvum 18S ssrRNA target sequence as described in Example 1 andmelting cure analysis was performed as described in Example 2. Theforward primers in Table 2 were used with reverse primer CryJVR:5′-ATTCCCCGTTACCCGTCA-3′ (SEQ ID NO: 5).

TABLE 2 Labeled target-specific primers SEQ # nt in G or C— Hairpin ID #loop position kcal/mol Sequence* Amplification melt 10 3 AGG-GG dG −14AGGCGCGATACGCGCCT GG ACTCA Yes Yes TAATAACTTTACGGAT 11 4 AGG-GG dG −14AGGCGCGATCACGCGCCT GG ACTC Yes Yes AGG- ATAATAACTTTACGGAT 12 5 GGGGdG −14 AGGCGCGATTCACGCGCCT GGGG A Yes Yes CTCATAATAACTTTACGGAT 13 3ACCC-GG dG −14 ACCCGCGATACGCGGGT GG ACTCA Yes No TAATAACTTTACGGAT 14 4ACCC-GG dG −14 ACCCGCGATACCGCGGGT GG ACTC Yes No ACCC- ATAATAACTTTACGGAT15 5 GGGG dG −14 ACCCGCGATAACCGCGGGT GGGG A Yes No CTCATAATAACTTTACGGAT16 3 ACC-GG dG −11 ACCGCGATACGCGGT GG ACTCATA Yes No ATAACTTTACGGAT 17 4ACC-GG dG −11 ACCGCGATCACGCGGT GG ACTCAT Yes No AATAACTTTACGGAT 18 5ACC-GG dG −11 ACCGCGATTCACGCGGT GG ACTCA Yes No TAATAACTTTACGGAT 19 3ACC-GG dG −7 ACCGCATAGCGGT GG ACTCATAAT Yes No AACTTTACGGAT 20 4 ACC-GGdG −7 ACCGCATCAGCGGT GG ACTCATAA Yes No TAACTTTACGGAT 21 5 ACC-GG dG −7ACCGCATTCAGCGGT GG ACTCATA Yes No ATAACTTTACGGAT 22 3 AGG-GG dG −11AGGCGCATAGCGCCT GG ACTCATA Yes Yes ATAACTTTACGGAT 23 3 AGG-GG dG −8AGGCGATACGCCT GG ACTCATAAT Yes Yes AACTTTACGGAT 24 4 AGG-GG dG −11AGGCGCATCAGCGCCT GG ACTCAT Yes Yes AATAACTTTACGGAT 25 4 AGG-GG dG −8AGGCGATCACGCCT GG ACTCATAA Yes Yes TAACTTTACGGAT 26 5 AGG-GG dG −11AGGCGCATTCAGCGCCT GG ACTCA Yes Yes TAATAACTTTACGGAT 27 5 AGG-GG dG −8AGGCGATTCACGCCT GG ACTCATA Yes Yes ATAACTTTACGGAT *Italicized lettersrepresent the stem-loop portion of the PET Tag (e.g., 16 of FIG. 2); Gsin bold are overhang nucleotides (e.g., 24 of FIG. 2); underlinedsequence is complimentary to the target DNA sequence (e.g., 30 of FIG.3); primers are labeled with FAM at the 5′-A.

As shown in Table 2, all of the primers were able to detect targetsequences using amplification. However, primers with 5′-ACC (instead of5′-AGG) at the 5′-end can also be used in amplification of target butdid not have the benefit of melting curve analysis. When thefluorescently labeled 5′-A comes into proximity to GG is quenchedinitially and quenching effect reduced when the complimentary CC's aresynthesized. Other parameters that influence the sensitivity of theassay included delta G (expressed as −kcal/mol) of the loop and numberof nucleotides in loop. In some examples, at least three nucleotides arerequired to form the loop of the stem loop structure. Although the loopsize can be increased, this is generally avoided to reduce productioncosts.

EXAMPLE 4 Effect of Additional G Nucleotides on 3′-End of Universal Tag

This example describes methods used to determine the effect onfluorescence on changing the number of Gs at the 3′-end of the PETprimer.

The PET tag 5′-FAM-AGGX₍₁₎X₍₂₎X₍₃₎ATAX₍₄₎X₍₅₎X₍₆₎CCTG(n) (SEQ ID NO: 28)was used to alter the number of Gs at G(n) on the 3′-end, wherein X₍₁₎is complementary to X₍₆₎, X₍₂₎ is complementary to X₍₅₎, and X₍₃₎ iscomplementary to X₍₄₎. In a specific example the PET tag was5′-FAM-AGGCGCATAGCGCCTX₍₁₎ (SEQ ID NO: 29), wherein X₍₁₎ is zero to twoG residues. The following primers were used:

#1: No 3′-end Gs, forward PET-tagged sequence specific primer was

SEQ ID NO: 30; 5′-FAM-AGGCGCATAGCGCCTATGACGGGTAACGGGGAAT;

#2—one 3′-end G, forward PET-tagged sequence specific primer was

SEQ ID NO: 31; 5′-FAM-AGGCGCATAGCGCCTGATGACGGGTAACGGGGAAT;and

#3—two 3′-end Gs, forward PET-tagged sequence specific primer was

SEQ ID NO: 32. 5′-FAM-AGGCGCATAGCGCCTGGATGACGGGTAACGGGGAAT;

The underlined portions of the PET primers are the targetsequence-specific primer sequences (non-underlined portion is the PETtag). All of these forward PET primers were used with reversesequence-specific primer CCAATTACAAAACCAAAAAGTCC (SEQ ID NO: 33) toamplify C. parvum DNA within the 18S ssrRNA gene as follows.

The target DNA was detected with the forward and reverse primersdescribed above. The forward primer was labeled with FAM at the 5′-end.DNA was extracted from C. parvum oocysts and suspended in 80 μl TrisEDTA (TE, pH 8.0) buffer. Two microliters of DNA were added perreaction. The amplification reaction mixture consisted of Quantifast®Probe PCR with no ROX vial kit reaction mixture (cat #204354—Qiagen),FAM-labeled PET forward primer and reverse primer (0.25 μM each). Analiquot (2 μl) of the extracted DNA sample was added to the PCR 96well-plate containing 18 μl reaction mixture along with appropriatenegative control were included in each experiment. The protocol tookapproximately 60 minutes to complete with the following PCR conditions:hot-start denaturation step at 95° C. for 3 minutes, followed by 45cycles with a 95° C. denaturation for 10 seconds, 60° C. annealing for50 seconds with single fluorescence acquisition in FAM™ dye, HEX™ dyeand Cy5® dye channels on a real-time PCR instrument (7500 Real-time PCRsystem). A positive result was recorded for FAM.

Traces for each of the three reactions with zero 3′-end Gs (#1), one3′-end G (#2), or two 3′-end Gs (#3) is shown in FIG. 9. As shown inFIG. 9, as the number of 3′-end G's increases from zero to two, the CTvalue decreases from approximately 38 (curve #1) to 34 (curve #2) to 31(curve #3). Thus the resulting amplicons can be detected at an earliercycle number. Even without a G at the 3′-end, there is still an increasein fluorescence due to the production of amplicons. This is becauseduring the hairpin folding stage the fluorescently-labeled nucleotide issandwiched between 2G's on either side and even if there are no G's onone side, the two G's on the other side quenches the fluorophore

EXAMPLE 5 Use of PET Primer in a Multiplex Format

This example describes methods used to demonstrate that PET primers canbe used in multiplex reactions.

The target DNA was detected with a forward and a reverse primer labeledeither with FAM or HEX at 5′-end, and a specific probe labeled withQuasar® 670 dye. DNA was extracted from C. parvum oocysts then suspendedin 80 μl Tris EDTA (TE, pH 8.0) buffer. Two microliters of DNA wereadded per reaction. The amplification reaction mixture consisted ofQuantifast® Probe PCR kit reaction mixture with no ROX (cat#204354—Qiagen), with one of the following primer/probe sets:

#1: FAM-labeled forward primer

(SEQ ID NO: 34) 5′-FAM-AGGCGGATACCGCCTGGATGACGGGTAACGGGGAAT,HEX-labeled reverse primer

(SEQ ID NO: 35) 5′-HEX-AGGCGGATACCGCCTGGCCAATTACAAAACCAAAAAGTCC(0.25 μM each) and Quas670 probe (0.2 μM)(Quas670-CGCGCCTGCTGCCTTCCTTAGATG-BHQ3; SEQ ID NO: 36); or

#2: HEX-labeled forward primer

(SEQ ID NO: 37) 5′-HEX-AGGCGGATACCGCCTGGATGACGGGTAACGGGGAAT,FAM-labeled reverse primer

(SEQ ID NO: 38) 5′-FAM-AGGCGGATACCGCCTGGCCAATTACAAAACCAAAAAGTCC(0.25 μM each) and Quas670 probe (0.2 μM; SEQ ID NO: 36)

The underlined portions of the PET primers are the targetsequence-specific primer sequences (non-underlined portion is the PETtag). An aliquot (2 μl) of the extracted DNA sample was added to the PCR96 well-plate containing 18 μl reaction mixture along with appropriatenegative control were included in each experiment. The protocol tookapproximately 30 minutes to complete with the following PCR conditions:hot-start denaturation step at 95° C. for 3 minutes, followed by 45cycles with a 95° C. denaturation for 10 seconds, 60° C. annealing for50 seconds with single fluorescence acquisition in FAM™ dye, HEX™ dyeand Cy5® dye channels on a real-time PCR instrument (7500 Real-time PCRsystem). A positive result was recorded for FAM™ dye, HEX™ dye and Cy5®dye channels.

As shown in FIGS. 10A and 10B, amplification of the targetCryptosporidium sequence was detected using PET forward and reverseprimers labeled with FAM (FIG. 10A) and HEX (FIG. 10B), respectively, asindicated by an increase in fluorescence over time. A TaqMan® probe wasincluded in the PET primer reactions to demonstrate that signal from alabeled probe could also be obtained in conjunction with use of the PETprimers. Fluorescence signal from a TaqMan® probe labeled with Quasar®670 dye at 5′-end and BHQ3 at the 3′-end (SEQ ID NO: 36) is shown inFIG. 10E. Similar PET primer (FIGS. 10C and 10D) and TaqMan® probe (FIG.10F) fluorescence results were obtained when the fluorophores wereswitched between the forward and reverse primers (FIGS. 10C and 10D).

EXAMPLE 6 Use of PET Tags with an Array

This example describes methods that can be used to detect the presenceof a nucleic acid molecule using the disclosed PET tags in combinationwith an array, such as a microarray.

In one example, the method includes amplification of a target nucleicacid sequence using a PET tag attached to the forward or reverse primer(e.g., see FIGS. 3A and B). The primer not containing a PET tag caninclude another label, such as a fluorophore, such as Cy3® or Cy5® dye.For example, real-time PCR can be performed using a forward primerlabeled with a PET tag using the methods disclosed herein, and a labeledreverse primer (for example labeled with Cy3® or Cy5® dye). Theresulting amplicons can be analyzed using the methods disclosed hereinto determine if the sample analyzed is positive or negative for thetarget nucleic acid of interest.

The resulting PCR products (amplicons) from the positive reactions canbe denatured at 100° C. for 2 minutes and chilled on ice immediatelyprior to hybridization to an array containing one or more nucleic acidsequence targets of interest. A particular example of such a microarrayis a DNA chip. In one example, the amino group of the target nucleicacid molecule can be linked at its 5′-end to the surface of the array.If the target nucleic acid sequence is present on the array, theamplicons previously generated (which contain at least one detectablelabel, such as two detectable labels) will hybridize to the targetnucleic acid on the array. The resulting hybridization will produce anincrease in signal due to the present of the detectable label on theamplicon. For example, if one of the primers included Cy3® dye or Cy5®dye, the resulting Cy3® dye or Cy5® dye labeled product will produce anincrease in fluorescence intensity, which can be detected and in someexamples further quantified.

Such a method can be used to confirm the positive or negative resultsobtained with amplification using a PET tag disclosed herein.

EXAMPLE 7 Use of Universal Tags with Pyrosequencing

This example describes methods that can be used to sequence a nucleicacid molecule using the disclosed PET tags in combination withpyrosequencing.

In one example, the method includes amplification of a target nucleicacid sequence using a PET tag attached to the 5′-end of a forward orreverse primer. The primer not containing a PET tag can include biotinat its 5′-end. The labeled forward and reverse primers are used toamplify a target nucleic acid sequence, for example by using real-timePCR methods disclosed herein. The resulting amplicons can be analyzedusing the methods disclosed herein to determine if the sample analyzedis positive or negative for the target nucleic acid of interest. Theresulting amplicons would contain a detectable biotin label.

The biotin labeled amplicon is separated after denaturation and adhesionof the amplicons to streptavidin-coated magnetic beads. The separatedstrands are then sequenced using pyrosequencing with an appropriatesequencing primer, using methods known in the art (for a review ofpyrosequencing see Franca et al., Q. Rev. Biophys. 35(2):169-200, 2002).

EXAMPLE 8 Detection of a Nucleic Acid Molecule in a Subject

This example describes methods to determine if a particular nucleic acidmolecule is present, for example present in a sample obtained from asubject.

In one example, the method includes amplification of a target nucleicacid sequence from a sample using a PET tag attached to the forward orreverse primer specific for the target nucleic acid sequence. In oneexample, the sample is obtained from a subject infected or suspected ofbeing infected with a pathogen, such as a virus, bacterium, parasite,fungi, or combinations thereof. In this example, the target nucleic acidsequence can be a sequence specific to the pathogen of interest, or anucleic acid molecule of the subject whose expression is altered (suchas increased or decreased) in response to the infection, or combinationsthereof.

In another example, the sample is obtained from a subject having orsuspected of having a disease, such as cancer. In particular examples,the subject is being treated or has been treated for the disease, andthe method is used to determine the subject's response to the treatment.In this example, the target nucleic acid sequence can be a nucleic acidmolecule of the subject whose expression is altered (such as increasedor decreased) due to the disease, a control sequence (such as a sequencethat detects expression of a housekeeping gene), or combinationsthereof. Housekeeping genes are known in the art (for example seeJanssens et al., Mol. Diagn. 8(2):107-13, 2004), and can includeporphobilinogen deaminase (PBGD); mitochondrial ATP synthase 6(mATPsy6); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).Ideally, a housekeeping gene has expression levels that remainrelatively constant in different experimental conditions.

The primer not containing a PET tag can include another label, such as afluorophore, such as Cy3® dye or Cy5® dye. For example, real-time PCRcan be performed using a forward primer labeled with a PET tag using themethods disclosed herein, and an unlabeled or labeled reverse primer(for example labeled with Cy3® dye or Cy5® dye). The resulting ampliconscan be analyzed using the methods disclosed herein to determine if thesample analyzed is positive or negative for the target nucleic acid ofinterest. If desired, the amplicons can be further analyzed, for exampleusing an array, to confirm the amplification results. In some examples,quantification of the target nucleic acid is performed.

In view of the many possible embodiments to which the principles of thedisclosure can be applied, it should be recognized that the illustratedembodiments are only examples of the disclosure and should not be takenas limiting the scope of the invention. Rather, the scope of thedisclosure is defined by the following claims. We therefore claim as ourinvention all that comes within the scope and spirit of these claims.

We claim:
 1. A method of making a labeled sequence-specific primer,comprising: adding a photoinduced electron transfer (PET) tag to asequence-specific primer, thereby generating a labeled sequence-specificprimer, wherein the PET tag comprises the nucleic acid sequence5′-X₁X_(2(a))X₃X_(4(a))G_(x)-3′ (SEQ ID NO: 1), wherein X₁ is a 5′-endlabeled nucleotide, wherein X₂ and X₄ comprise a stem of a stem-loop andare nucleotide sequences of length a, wherein a is 3 or more nucleotidesand wherein X₂ is at least 60% complementary to X₄, wherein X₃ comprisesa loop of the stem-loop, and wherein G_(x) comprises at least twoconsecutive G nucleotides; wherein the stem-loop brings the label on the5′-end-labeled nucleotide and the at least two consecutive G nucleotidesinto proximity, thereby quenching a detectable signal from the5′-end-labeled nucleotide in the absence of a target nucleic acidsequence; wherein the sequence-specific primer can hybridize to thetarget nucleic acid sequence; wherein the PET tag does not substantiallyhybridize to the target nucleic acid sequence recognized by thesequence-specific primer; and wherein the detectable signal from the5′-end-labeled nucleotide is unquenched when the labeledsequence-specific primer is incorporated into an amplicon.
 2. The methodof claim 1, wherein the PET tag comprises the sequence5′-X₁X_(2(a))X₃X_(4(a))G_(x)X_(5(n))-3′ (SEQ ID NO: 2), wherein X_(5(n))comprises one or more nucleotides.
 3. The method of claim 1, wherein X₁is not G.
 4. The method of claim 1, wherein X₂ has at least 80%complementarity to X₄.
 5. The method of claim 4, wherein X₂ is 100%complementarity to X₄.
 6. The method of claim 1, wherein X₃ is 3 or morenucleotides.
 7. The method of claim 1, wherein X₃ is a trinucleotidesequence selected from the group consisting of TAA, ATA, AAT, TTA, TAT,ATT, TTT and AAA.
 8. The method of claim 1, wherein X₃ does not includeC or G nucleotides.
 9. The method of claim 1, wherein the label is afluorophore.
 10. The method of claim 1, wherein the PET tag is 12 to 20nucleotides in length.
 11. The method of claim 1, wherein a 5′-end ofthe sequence-specific primer is attached to the 3′-end of the PET tag.12. A labeled sequence-specific primer generated using the method ofclaim
 1. 13. A kit comprising: the labeled sequence-specific primer ofclaim 12; and a buffer.
 14. A method of detecting a target nucleic acidmolecule comprising: incubating a sample comprising the target nucleicacid molecule with a forward primer comprising a sequence homologous tothe target nucleic acid molecule and a reverse primer comprising asequence homologous to the target nucleic acid molecule under conditionssufficient to allow amplification of the target nucleic acid molecule;amplifying the target nucleic acid molecule using real-time polymerasechain reaction (PCR), thereby generating a labeled amplicon; denaturingthe labeled amplicon; generating a melting curve; and detecting a signalfrom the label, wherein the forward primer or the reverse primer islinked at its 5′-end to the 3′-end of a photoinduced electron transfer(PET) tag, wherein the PET tag comprises the sequence5′-X₁X_(2(a))X₃X_(4(a))G_(x)-3′ (SEQ ID NO: 1), wherein X₁ is a 5′-endlabeled nucleotide, wherein X₂ and X₄ comprise a stem of a stem-loop andare nucleotide sequences of length a, wherein a is 3 or more nucleotidesand wherein X₂ is at least 60% complementary to X₄, wherein X₃ comprisesa loop of the stem-loop, and wherein G_(x) comprises at least twoconsecutive G nucleotides, wherein the stem-loop brings the label on the5′-end-labeled nucleotide and the at least two consecutive G nucleotidesinto proximity, thereby quenching a detectable signal from the5′-end-labeled nucleotide in the absence of a target nucleic acidsequence, and wherein the PET tag does not substantially hybridize tothe target nucleic acid sequence recognized by the forward and reverseprimers, and wherein the detectable signal from the 5′-end-labelednucleotide is unquenched when the labeled forward or reverse primer isincorporated into the labeled amplicon, wherein an increase in signaldetected during the real-time PCR indicates that the target nucleic acidmolecule is present in the sample and wherein no significant increase insignal detected during the real-time PCR indicates that the targetmolecule is not present in the sample, and wherein a single peakdetected during generating the melting curve indicates that the targetnucleic acid molecule is present in the sample and wherein no singlepeak detected during generating the melting curve indicates that thetarget molecule is not present in the sample.
 15. The method of claim14, further comprising quantifying the signal from the label.
 16. Themethod of claim 14, wherein the forward primer is linked at its 5′-endto the 3′-end portion of the PET tag and wherein the reverse primer isnot linked to the PET tag.
 17. The method of claim 14, wherein thereverse primer is linked at its 5′-end to the 3′-end portion of the PETtag and wherein the forward primer is not linked to the PET tag.
 18. Amethod of detecting a polymorphism in a target nucleic acid moleculecomprising: incubating a sample comprising the target nucleic acidmolecule with a forward primer and a reverse primer, wherein the forwardprimer or the reverse primer is linked at its 5′-end to the 3′-end of aPET tag under conditions sufficient to allow amplification of the targetnucleic acid molecule, thereby generating a labeled amplicon, whereinthe PET tag comprises the sequence 5′-X₁X_(2(a))X₃X_(4(a))G_(x)-3′ (SEQID NO: 1), wherein X₁ is a 5′-end labeled nucleotide, wherein X₂ and X₄comprise a stem of a stem-loop and are nucleotide sequences of length a,wherein a is 3 or more nucleotides and wherein X₂ is at least 60%complementary to X₄, wherein X₃ comprises a loop of the stem-loop, andwherein G_(x) comprises at least two consecutive G nucleotides, whereinthe stem-loop brings the label on the 5′-end-labeled nucleotide and theat least two consecutive G nucleotides into proximity, thereby quenchinga detectable signal from the 5′-end-labeled nucleotide in the absence ofa target nucleic acid sequence, and wherein the PET nucleic acidsequence does not substantially hybridize to the target nucleic acidsequence recognized by the forward and reverse primers, and wherein thedetectable signal from the 5′-end-labeled nucleotide is unquenched whenthe labeled forward or reverse primer is incorporated into an amplicon;and detecting a change in signal from the label while exposing thelabeled amplicon to conditions that permit denaturation of the ampliconinto single-stranded nucleic acid molecules, wherein the change insignal is directly proportional to the extent of amplicon denaturation,and wherein differences in the extent of amplicon denaturation representa polymorphism in the target nucleic acid.
 19. The method of claim 18,wherein the forward primer is linked at its 5′-end to the 3′-end of thePET tag and wherein the reverse primer is not linked to the PET tag. 20.The method of claim 18, wherein the reverse primer is linked at its5′-end to the 3′-end of the PET tag and wherein the forward primer isnot linked to the PET tag.